Abstract
Microtubule-dependent movement is crucial for the spatial organization of endosomes in most eukaryotes, but as yet there has been no systematic analysis of how a particular microtubule motor contributes to early endosome dynamics. Here we tracked early endosomes labeled with GFP-Rab5 on the nanometer scale, and combined this with global, first passage probability (FPP) analysis to provide an unbiased description of how the minus-end microtubule motor, cytoplasmic dynein, supports endosome motility. Dynein contributes to short-range endosome movement, but in particular drives 85–98% of long, inward translocations. For these, it requires an intact dynactin complex to allow membrane-bound p150Glued to activate dynein, since p50 over-expression, which disrupts the dynactin complex, inhibits inward movement even though dynein and p150Glued remain membrane-bound. Long dynein-dependent movements occur via bursts at up to ∼8 µms−1 that are linked by changes in rate or pauses. These peak speeds during rapid inward endosome movement are still seen when cellular dynein levels are 50-fold reduced by RNAi knock-down of dynein heavy chain, while the number of movements is reduced 5-fold. Altogether, these findings identify how dynein helps define the dynamics of early endosomes.
Highlights
The motility of organelles within the endocytic pathway contributes to the passage and sorting of endocytosed material [1,2,3], as well to the spatial organization of endosomal signalling platforms [4,5]
This motility is powered by the activity of motor proteins that drive endosome movement along microtubules and/or actin [6]
Endosomes interact with many motor proteins, including the minus-end microtubule motor dynein [7,8,9,10,11,12,13,14], minus-end kinesins such as KIFC1 [15] and KIFC2 [16], and plus-end kinesins including kinesin-1 [8,15,17] and kinesin-3 family members such as KIF16B [18] and others [10,19]
Summary
The motility of organelles within the endocytic pathway contributes to the passage and sorting of endocytosed material [1,2,3], as well to the spatial organization of endosomal signalling platforms [4,5]. Dynein drives both short and long range early endosome motility We have previously identified dynein as the motor that drives the inward movement of EGF-containing early endosomes in HeLa cells, but this analysis was performed at low temporal resolution (1 frame s21: [7]).
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